The C-terminal domain (CTD) of RNA polymerase II (RNAPII) plays a pivotal role in orchestrating RNA processing and other co-transcriptional events to achieve proper gene expression. It consists of multiple heptad repeats (Y1S2P3T4S5P6S7) that are highly conserved from yeast to human. The predominant form of CTD modification is the phosphorylation of Ser2 and/or Ser5 within the heptad repeats. The level and pattern of CTD phosphorylation are regulated by the concerted action of CTD kinases and phosphatases during the transcription cycle. The vast number of CTD phosphorylation states, also known as the "CTD code," form the basis for recruitment of specific macromolecular complexes to the transcribing polymerase. Recent identification of novel phosphoCTD-associating proteins has expanded the known functions of the CTD from mediating RNA processing to coordinating other co-transcriptional events, such as chromatin remodeling. Compared to the rapid progress in CTD biology, the structural knowledge of CTD recognition by CTD- associating proteins and CTD-modifying enzymes is very limited. Our long term goal is to understand the structures and mechanisms of CTD-modifying enzymes and CTD-mediated assembly of co-transcriptional complexes. In this proposal, we describe structural, biochemical and functional studies to investigate (1) the structure of the SRI domain of the Set2 histone methyltransferase and its interaction with the phosphoCTD;(2) the interactions between tandem FF domains of CA150 and the phosphoCTD;(3) the structure and mechanism of a novel Ser5-specific CTD phosphatase, Ssu72. PUBLIC HEALTH RELEVANCE (Public Health Relevance Statement) Abnormal changes of CTD phosphorylation states and their associated processes have been connected to a variety of human diseases. A number of viruses, including HIV-1, have developed effective means to modulate the CTD phosphorylation pattern or recruit CTD-associating nuclear factors in order to enhance viral gene expression. Advances in our structural understanding of (phospho)CTD-associating proteins, their interactions with the (phospho)CTD, and CTD-modifying enzymes are likely to generate a significant impact on issues related to human disease and ultimately contribute to improving human health.